1. Field of the Invention
This invention relates to improved methods for detecting mutations in nucleic acid sequences and, specifically, to the detection of single base change mutations. More particularly, the invention concerns the use of improved reaction conditions, such as concomitantly decreased salt and RNase enzyme concentrations, that allow the detection of many single base mutations which were undetectable in the previously known reaction conditions.
2. Description of the Related Art
Methods for easily and reliably detecting point mutations without prior knowledge of the exact location of the mutations, have wide application in diagnosis and treatment of genetic diseases and cancers, as well as use in genetic counseling. These methods also are of great benefit in the analysis of a variety of human genetic diseases and in establishing human genetic linkage maps.
In many genetic diseases, the causative mutations are scattered over a large number of sites. For example in retinoblastoma, 30% of the cases are the result of widely scattered new mutations (Yandell et al., 1989). In addition, accumulation of point mutations in a variety of genes, such as, for example, p53, ras, and other "protooncogenes" are thought to play fundamental roles in the multi-step process of transformation of normal cells to the malignant state. The ability to detect these mutations is important both for genetic counseling and for early clinical intervention. Improved efficiency and reliability in methods of detecting point mutations should lead to a better understanding of the mechanisms of carcinogenesis and to improved treatment and prognosis for a variety of cancers. The ideal screening method would quickly, inexpensively, and reliably detect all types of widely dispersed point mutations, insertions/deletions, and translocations in genomic DNA, cDNA, or RNA samples depending on the specific situation. Currently there are no methods which achieve these goals.
Over the past ten years, a number of different methods have been used to detect single-base mutations. These methods include denaturing gradient gel electrophoresis, restriction enzyme polymorphism analysis, chemical mismatch methods and others (see Cotton, 1989, for a review of single-base mutation detection methods). Recently, SSCP (single-strand conformation polymorphism) analysis and the closely related heteroduplex analysis methods have come into use for screening for single-base mutations (Orita et al., 1989; Keen et al., 1991). In these methods, the mobility of PCR-amplified test DNA from clinical specimens is compared with the mobility of DNA amplified from normal sources by direct electrophoresis of samples in adjacent lanes of native polyacrylamide or other types of matrix gels. Single-base mutations often alter the secondary structure of the molecule sufficiently to cause slight mobility differences between the normal and mutant PCR products after prolonged electrophoresis.
Unfortunately, SSCP has several major drawbacks. The most important is that not all mutations result in detectable shifts in mobility. Recently it was found that of 20 mutations detected by direct sequencing, only 35% were detected by SSCP (Sarkar et al., 1992). Other studies have reported higher detection efficiencies, but it is clear that SSCP has a major problem in missing point mutations. Chances of detecting mobility differences can be increased by running parallel gels under different conditions, for example at 4.degree. C. and 30.degree. C., with and without 5% glycerol, (Hayashi, 1991), but this significantly increases the cost and labor associated with analysis. Since mobility differences are generally quite small, analysis of genes in the heterozygous state is compromised. Another drawback of SSCP and related techniques is that they provide no information on the position of the mutation within the DNA fragment being analyzed. Also, the time required for SSCP-type analysis is fairly long, since electrophoresis often requires 12-24 hours to resolve the fragments. Furthermore, there seems to be an upper size limit for analysis by SSCP of approximately 300 bases and increased fragment length has been associated with decreased efficiency of mutation detection (Hayashi, 1991).
Direct sequencing of PCR products is often considered to be the most reliable method of identifying unknown mutations. However, the labor and time involved in direct sequencing are extensive. In fact, direct sequencing is the most time consuming step in the identification of point mutations even with the availability of automated sequencing methods. Further, even DNA sequencing may not give a clear indication of a single-base mutation when an individual is heterozygous for that allele. The ambiguity arises because the resulting co-incident bands at the relevant position on the sequencing ladder (Cheng and Haas, 1992) could be mistaken for the ubiquitous artifact of "shadow bands", which are caused by premature termination during the extension reaction. Therefore, the development of reliable, preliminary screening methods to eliminate the unnecessary sequencing of DNA fragments which do not contain mutations is an immediate need in the art.
Ribonuclease protection assay (RPA) is another technique used for detection of dispersed single-base mutations. In this procedure, a labeled antisense RNA probe is hybridized to a complementary test RNA or DNA in solution, and then the remaining unhybridized, single-stranded probe is degraded by ribonuclease treatment. The hybridized, double stranded probe is protected from RNAse digestion. After an appropriate time, the products of the digestion reaction are recovered and analyzed on a gel. If there is a single-base mismatch between the complementary probe and the test nucleic acids, the ribonuclease may cleave the probe at that position, resulting in the appearance of two new bands on the gel (Winter et al., 1985; Myers et al., 1985; Perucho, 1989). The size of these protected fragments gives information regarding the location of the mutation. RPAs have been used to detect single-base mismatches in many different genes, including p53, ras, myc, retinoblastoma, APC, HIV reverse transcriptase, .beta. globin and others (Takahashi, et al., 1989; Forrester et al., 1987; Richman and Hayday, 1989; Dunn et al., 1988; Lopez-Galindez et al., 1991; Myers et al., 1985; Winter et al., 1985).
Historically, RPAs have been successful in detecting about half of all point mutations, provided the analysis is performed on both strands of the test DNA (Myers and Maniatis, 1986; Kinzler et al., 1991). However, some detectable mismatches are only partially cleaved, which decreases the sensitivity of the technique and complicates analysis of heterozygous mutations. The ability to detect all point mutations would greatly increase the utility of this technique and enable it to be applied in clinical settings where a greater than 50% detection rate is necessary.